Chop up DNA into small pieces using an enzyme that recognizes specific DNA sequences and cleaves DNA at these sequences. Consult the New England Biolabs catalog, as it is the best resource for these experiments.
- Starting DNA should be pure and dissolved in water.
- Amount of DNA will depend on what you will be doing with the restriction fragments.
- Concentration should be at least 0.2 µg/µl.
- For Southern Blots, generally need from 5 to 10 µg of DNA.
- Enzyme: Use the New England Biolabs enzyme charts to determine what enzyme and buffers to use in the restriction digest
- Perform all steps on ice
- Amount of enzyme to use. Generally 1 µl of enzyme (= 5 units) per µg of DNA. The # of units depends on cleavage rate of DNA substrate. NEB uses "cohesive end units" while invitrogen uses Weiss units. 1 weiss unit = 67 coehsive end units. 1 cohesive end unit = 0.015 weiss units. So Invitrogen 1 U/µl = NEB 67 U/µl. Supercoiled plasmids and agarose-embedded DNAs usually require more than 1 unit/µg to be cleaved completely. 1 unit is generally defined as the amount of enzyme required to completely digest 1 µg DNA in 50 µl in 60 min @ 37°C. Generally, use 5-10 fold excess in reaction: for 5 µg of DNA, need 25-50 Units (i.e. 0.5-1.0 Units/µl in 50 µl)
- Reaction amounts: Generally digest 1 µg in total volume of 40 µl. Use the Restriction Digest excel spreadsheet to calculate amounts of DNA, restriction enzyme, buffers, and H2O to add to each tube.
- Label the tubes and thaw the DNA.
- To ensure better digestion, let reaction (without restriction enzyme) sit at 4°C for several hours to disperse any DNA clumps
- Add Restriction enzyme last. Gently pipette or flick tube, brief spin-down. Do not vortex.
- Incubate for 1 hour to overnight at 37°C (unless using special high temp. restriction enzyme).
- Stop reaction: Can add stop solution (50% glycerol, 50 mM EDTA (pH8.0), and 0.05% bromophenol blue (10 µl/50 µl rxn). Or raise temp to 65 or 80°C for 20 min (read the NEB catalog to see what temperature is appropriate for the specific restriction enzyme. Or perform phenol chloroform extraction.
- Include a control reaction with no Restriction enzyme (degradation of DNA indicates contamination in DNA prep or buffers). Control DNA (lambda or adenovirus-2 DNA). Mix control with experimental DNA to determine if inhibitor is present in experimental sample (e.g. salt, EDTA or phenol).